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Heterolytic OO bond cleavage: Functional role of Glu113 during bis-Fe(IV) formation in MauG.

ABSTRACT: The diheme enzyme MauG utilizes H2O2 to perform oxidative posttranslational modification on a protein substrate. A bis-Fe(IV) species of MauG was previously identified as a key intermediate in this reaction. Heterolytic cleavage of the OO bond of H2O2 drives the formation of the bis-Fe(IV) intermediate. In this work, we tested a hypothesis that a glutamate residue, Glu113 in the distal pocket of the pentacoordinate heme of MauG, facilitates heterolytic OO bond cleavage, thereby leading to bis-Fe(IV) formation. This hypothesis was proposed based on sequence alignment and structural comparison with other H2O2-utilizing hemoenzymes, especially those from the diheme enzyme superfamily that MauG belongs to. Electron paramagnetic resonance (EPR) characterization of the reaction between MauG and H2O2 revealed that mutation of Glu113 inhibited heterolytic OO bond cleavage, in agreement with our hypothesis. This result was further confirmed by the HPLC study in which an analog of H2O2, cumene hydroperoxide, was used to probe the pattern of OO bond cleavage. Together, our data suggest that Glu113 functions as an acid-base catalyst to assist heterolytic OO bond cleavage during the early stage of the catalytic reaction. This work advances our mechanistic understanding of the H2O2-activation process during bis-Fe(IV) formation in MauG.

SUBMITTER: Geng J

PROVIDER: S-EPMC5191938 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Heterolytic OO bond cleavage: Functional role of Glu113 during bis-Fe(IV) formation in MauG.

Geng Jiafeng J Huo Lu L Liu Aimin A

Journal of inorganic biochemistry 20161109

The diheme enzyme MauG utilizes H2O2 to perform oxidative posttranslational modification on a protein substrate. A bis-Fe(IV) species of MauG was previously identified as a key intermediate in this reaction. Heterolytic cleavage of the OO bond of H2O2 drives the formation of the bis-Fe(IV) intermediate. In this work, we tested a hypothesis that a glutamate residue, Glu113 in the distal pocket of the pentacoordinate heme of MauG, facilitates heterolytic ...[more]

PMID: 27907864

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Project description:Heme hydroperoxidases catalyze the oxidation of substrates by H2O2. The catalytic cycle involves the formation of a highly oxidizing species known as Compound I, resulting from the two-electron oxidation of the ferric heme in the active site of the resting enzyme. This high-valent intermediate is formed upon facile heterolysis of the O-O bond in the initial FeIII-OOH complex. Heterolysis is assisted by the histidine and arginine residues present in the heme distal cavity. This chemistry has not been successfully modeled in synthetic systems up to now. In this work, we have used a series of iron(iii) porphyrin complexes (FeIIIL2(Br), FeIIIL3(Br) and FeIIIMPh(Br)) with covalently attached pendent basic groups (pyridine and primary amine) mimicking the histidine and arginine residues in the distal-pocket of natural heme enzymes. The presence of pendent basic groups, capable of 2nd sphere hydrogen bonding interactions, leads to almost 1000-fold enhancement in the rate of Compound I formation from peracids relative to analogous complexes without these residues. The short-lived Compound I intermediate formed at cryogenic temperatures could be detected using UV-vis electronic absorption spectroscopy and also trapped to be unequivocally identified by 9 GHz EPR spectroscopy at 4 K. The broad (2000 G) and axial EPR spectrum of an exchange-coupled oxoferryl-porphyrin radical species, [FeIV[double bond, length as m-dash]O Por˙+] with g eff ⊥ = 3.80 and g eff ‖ = 1.99, was observed upon a reaction of the FeIIIL3(Br) porphyrin complex with m-CPBA. The characterization of the reactivity of the FeIII porphyrin complexes with a substrate in the presence of an oxidant like m-CPBA by UV-vis electronic absorption spectroscopy showed that they are capable of oxidizing two equivalents of inorganic and organic substrate(s) like ferrocene, 2,4,6-tritertiary butyl phenol and o-phenylenediamine. These oxidations are catalytic with a turnover number (TON) as high as 350. Density Functional Theory (DFT) calculations show that the mechanism of O-O bond activation by 2nd sphere hydrogen bonding interaction from these pendent basic groups, which are protonated by a peracid, involves polarization of the O-O σ-bond, leading to lowering of the O-O σ*-orbital allowing enhanced back bonding from the iron center. These results demonstrate how inclusion of 2nd sphere hydrogen bonding interaction can play a critical role in O-O bond heterolysis.

Dataset Information

Heterolytic OO bond cleavage: Functional role of Glu113 during bis-Fe(IV) formation in MauG.

Publications

Heterolytic OO bond cleavage: Functional role of Glu113 during bis-Fe(IV) formation in MauG.

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